1. Field of the Invention
The invention relates to methodologies for analyzing the mass spectrum of macromolecules from ionization sources and in particular to a methodology directed to enhancing the mass spectrum by emphasizing molecular peaks distributed across a plurality of charged states.
2. Description of the Prior Art
Mass spectroscopy is a tool used for characterizing macromolecules. Recently, technologies have been developed for matrix assisted laser desorption and electrospray ionization (ESI) methods for introducing samples into the mass spectrum analyzers. These new techniques allow very high molecular weight species to be introduced into a gas phase and ionized without significant degradation of the macromolecule. Electrospray ionization also has the advantage of having a very high ionization efficiency, which allows useful spectra required for even very small quantities of material. See R. D. Smith et al. Anal. Chem. 1990, 62, 882-99. Additionally, an ESI source is able to deposit multiple charges on a single molecule in a very reproducible manner. See Smith supra, M. Mann et al. Anal. Chem. 1989. 61, 1702-8 and J. A. Loo et al., Anal. Chem. 1991, 63, 2488-99.
The ability to put multiple charges on a molecule allows a conventional mugs analyzer with a limited range of mass-to-charge ratios to record a spectrum for very high molecular weight ions. In the extreme, spectra of molecules with molecular weights as large as 5 10.sup.6 Daltons have been recorded. See T. Nohmi et al., J. Am. Chem. Soc. 1992, 114, 3241. In addition, when a molecule carries multiple charges, the distribution of charges on the molecule can often be observed. The multiplicity of charge states gives rise to an envelope of peaks in the mass-to-charge spectrum produced by the analyzer. Although the envelope of the spectrum may appear to be complicated, in practice the peaks provide a means by which the mass of the ion can be determined.
However, the use ESI in mass spectrometers has been impeded by a lack of robustness in the methods for determining the mass of the molecule. Many peaks are exhibited in the output of the mass spectrometer such as is typically shown in Fenn et al, "Method of Producing Multiply Charged Ions and for Determining Molecular Weights of Molecules by the Use of Multiply Charged Ions of Molecules," U.S. Pat. No. 5,130,538 (1992), which is incorporated herein by reference.
One methodology, which has addressed the problem of analyzing the peaks to produce a calculated or enhanced output spectrum with greater robustness, is described by Mann, et al., supra. Mann utilizes a methodology based upon the mass-to-charge ratio of peaks in the electrospray spectrum designated as M.sub.n. The n designates the number of charges on the ion and M.sub.p is the mass of the parent ion. The mass, m.sub.c, is the mass of the counter ion which carries the charge such as a proton, sodium or potassium ion adducted to the molecule from a water solution. Equation 1 below illustrates relationship between these quantities. EQU M.sub.n =M.sub.p /n+m.sub.c ( 1)
In the first methodology described by Mann, there is a requirement that the mass-to-charge ratio, M.sub.n, for two peaks in the electrospray spectrum, the charge difference between these two peaks, and the mass of counter ion must be known in order for the methodology to be performed. Given this information, two expressions based upon Equation 1 above can be established and solved to determine the mass of the parent ion M.sub.p which is the object of the methodology.
The difficultly with this prior art methodology is the peak selection process. The information which is required to be known for the methodology, to be used is often difficult to extract from the spectrum produced by the mass analyzer, if there is a poor signal-to-noise ratio or, if the spectrum contains signals from multiple parent ions, i.e. the sample has more than one type of substance in it.
Mann has also proposed a second methodology for use in electrospray spectra by assuming an additive correlation between the peaks in the spectrum. According to the second methodology, the sum of the intensities that each of the peaks in the mass spectrum at integral fractions of a chosen mass is calculated to produce an enhanced intensity. This calculation is repeated over a selected mass range to generate an enhanced mass spectrum.
Although the algorithm is easily implemented, the method generates spurious peaks in the calculated or enhanced spectrum when the selected mass used for the calculation does not correspond to a molecular ion but has a partial correlation with the empirically measured peak envelope. This causes false peaks to be generated which often approach the intensity of the true molecular ion peaks. The result is that the enhanced spectrum is not necessarily easier to interpret than the measured spectrum itself.
More recently, Reinhold and Reinhold describe a methodology which uses maximum entropy to estimate an enhanced mass spectrum from an electrospray mass-to-charge spectrum. See B. B. Reinhold et al., J. Am. Soc. Mass Spectrom. 1992, 3, 207-15. According to the Reinhold methodology, a molecular weight is assumed and the electrospray spectrum is predicted for the assumed mass of the molecule. The difference between the estimated and actual spectrums are calculated and then the difference is plotted as a function of mass over a range selected by the user. The maximum entropy calculation provides added discrimination against artifact peaks generated by the Mann deconvolution methodology, but the calculations in the Reinhold system are slow and a priori knowledge of the peak shapes and relative intensities in the mass-to-charge spectrum are required if there is to be optimal processing of the data.
What is needed then is a methodology for analyzing the mass spectrum of a multiply charged macromolecule which makes better use of the information contained within the electrospray spectrum.
Specifically, what is needed is a methodology which can make use of the fact that electrospray ionization usually distributes an ion signal between two or more charged states on the molecule. Thus, peaks which might arise from the low molecular weight species and the partial correlation between these peaks and the envelope are suppressed relative to the true molecular peaks.
What is needed is a practical approach in which the enhanced mass spectrum of the molecule is easier to interpret than the enhanced spectrums generated by the prior art Mann or Reinhold deconvolution methodologies.